Process and device for the isolation of cell components, such as nucleic acids, from natural sources

ABSTRACT

A filtration process for the preparation of nucleic acids from natural sources is disclosed. The sources containing nucleic acids are lysed; the lysate is left to rest for some time; the resulting lysate passes a filter layer. The filter layer is selected from silica gel, aluminum oxide or packed diatomaceous earth, or interwoven or cemented non-wovens of glass fibers and silica gel. Other filter layers include cellulose, paper, compressed paper, and paper non-wovens. The fraction leaving the filter layer is then collected and the nucleic acid is subsequently isolated and purified from the collected fraction. Furthermore, the filter layer has not been modified with anion exchange groups.

This application is a 371 of PCT/EP95/00392 filed Feb. 3, 1995.

This invention is directed to a process for the preparation of nucleicacids from natural sources, such as E. coli cells, human and animalcells, by separating the lysed natural sources, such as cells or celldebris, in a sample by filtration, and to a kit containing means forperforming said process.

Frequently, in the preparation of cell components, particularly nucleicacids, the problem arises to separate the lysed natural sources, fromwhich the components are derived, from dissolved material. Usually, theseparation of cells or cell debris is effected by centrifugation,whereby cell fragments or cells deposit as a pellet in the centrifugetube. The dissolved cell components then are found in the supernatantand may be pipetted. In the preparation of nucleic acids, simplefiltration methods were not successful for the separation of the lysedcells or their fragments because the cell fragments either pass throughthe filter having too large a pore size and thus give rise to turbidityand impurities in the filtrate or, when filters with appropriatelynarrow pores are used, inevitable jamming will result, so that apurposeful preparation of the cell components is no longer possible.

Thus, the present invention is based on the problem of providing aprocess and creating a device by means of which centrifugation steps forthe preparation of cell components from natural sources, such as cells,may be avoided by using filtration steps which are easier to handle.

Conventionally, in order to isolate components from cells, the latterare lysed first. In the preparation of nucleic acids, the cells have tobe lysed first, for instance, by using enzymes, such as e.g. proteinaseK and lysozyme, detergents, such as SDS, Brij, Triton X 100, Tween 10,and DOC, and chemicals, such as sodium hydroxide, guanidinehydrochloride and guanidine isothiocyanate. The cell fragments aresedimented by centrifugation, and the supernatant is decanted orpipetted off and subsequently purified by chromatography or extractionwith phenol or chloroform and an alcohol precipitation. Maniatis;Current Protocols in Molecular Biology, Ausubel, F. M. et al., eds.(1991), Wiley Interscience, New York; Birnboim H. C. and Doly, J.(1979), A Rapid Alkaline Extraction Procedure for Screening RecombinantPlasmid DNA. Nucl. Acids Res. 7, pp 1513-1522.

Such centrifugation in a conventional laboratory centrifuge, e.g.,Heraeus Biofuge GL, Beckmann GS6, will take from 15 min to 2 h at from3,000 rpm to 20,000 rpm, depending on the particular application and theparticle size of the cell fragments. This means that with large samplenumbers, the centrifugation will require much personnel and involvelosses in time. Therefore, it is desirable to be provided with a simpleand rapid process for the removal of the cell fragments.

In preliminary experiments, the filter materials and filtration methodswhich have been available to date have proven to be unsuited for theseparation of such biological cell debris. Sterile filters, e.g. made ofnylon or cellulose acetate, having pore sizes of from 0.2 μm or 0.45 μmwill instantly clog and lack the capacity of retaining a larger amountof cell fragments.

These filters are only useful for the filtration of liquids having avery low content of solids or cellular contaminations.

In the filtration process according to the invention for the preparationof nucleic acid from natural sources, the sources containing nucleicacids are first lysed. The lysate is left to rest for some time.Preferably, the resting time is at least 1 minute, from 5 to 10 minutesbeing particularly preferred. The resulting lysate then passes a filterlayer of glass, silica gel, titanium oxide, aluminium oxide or packeddiatomaceous earth, or interwoven or cemented non-wovens of glass fibersand silica gel, as well as cellulose, paper, compressed paper, papernon-wovens. Thereafter, the fraction leaving the filter layer iscollected and the nucleic acid is subsequently isolated and purifiedfrom the collected fraction.

Especially with a packed filter layer of silica gel having a particlesize of from 15 to 30 μm, cell fragments can be retained successfullyand without any clogging of the filter, and a clear lysate can beobtained.

Preferably, the filter layers are modified such that there is noaffinity for nucleic acids. In particular, minerals bearing hydroxygroups or coated materials, such as diol-silica gel, diol-diatomaceousearth, and/or diol-perlite, may be used.

In a preferred embodiment, sample flow through the filter layer may befacilitated by applying positive or negative pressure. However, due tothe pore size configuration of the filter layer, passage of the sampleto be filtrated through the filter layer is also possible driven bygravity. Furthermore, in order to accelerate the passage of samplethrough the filter layer, the sample may also be passed through thefilter layer by centrifugation.

As the filter layers, there are used, e.g., silica gel, glass ordiatomaceous earth having particle sizes of from 5 μm to 100 μm.

A particularly preferred filter layer is untreated diatomaceous earth ormodified diol-diatomaceous earth having flow values of from 0.1 to 15Darcy.

The sources containing nucleic acids may be cells from cell cultures,any kind of tissues, body fluids or microorganisms.

The process according to the invention is particularly suitable for thepreparation of plasmid DNA from E. coli, yeast or eukaryontic cells, orof genomic DNA from blood or cells, the DNA having a size of from 1 to50 kb. The process according to the invention is also useful forpurifying plasmid DNA, cosmid DNA, in particular formolecular-biological research, such as cloning and sequencing, plasmidDNA for gene therapy, genomic DNA for analytics, diagnostics and genetherapy, and/or viral nucleic acids.

The positive pressure at the side before the passage through the filterlayer is preferably achieved by means of a piston.

Preferably, the filtration is followed by further processing steps, suchas separation on anion exchangers and/or adsorption to and desorptionfrom other mineral supports.

A device for performing the process consists of a hollow body in whichthe filter layer is arranged. The filter layer is preferably arrangedbetween two fixing means. As said hollow body, there may be used asyringe, in particular.

As the filter layer which may be used in the process according to theinvention, there may be used layers in the form of packings, inparticular of glass, silica gel, titanium oxide, alumina, ordiatomaceous earth, e.g. cellite or silica gel or perlite, but alsointerwoven or cemented non-wovens of glass fibers and silica, as well aspaper, compressed paper, paper non-wovens, or combinations thereof.Other particles suitable for filtration made of minerals or syntheticpolymers, diatomaceous earth, silica gel, perlite, and other mineralsupports are either untreated or treated to have a hydrophilic surfacewhich is hardly capable of adsorbing nucleic acids, e.g. diol modifieddiatomaceous earth.

In a preferred embodiment of the process according to the invention,multiple samples are processed simultaneously and passed throughappropriate devices advantageously adapted to microtitration systems.

A suitable device for performing the process according to the inventionconsists of a preferably cylindrical hollow body having an inlet andoutlet and a filtration means arranged in the hollow body. For securingthe filtration means, common securing means may be used, such as, forexample, cementings, but also securing by frictional forces by jammingthe filtration means in the hollow body.

BRIEF DESCRIPTION OF THE DRAWING

The device consists of at least one filter layer with equal pore sizesas seen in the direction of outlet 60. FIG. 1 shows a particularlypreferred variant of the device, where in the preferably cylindricalhollow body 40, the filtration means 70 is designed from one sheet. Theparticle size of the filter layer is in the range of from 5 μm to 500 μmat a total thickness of the filter layer of from 0.1 to 200 mm.

It may be advantageous to arrange an additional layer 23 in the hollowbody 40, namely, above and/or below layer 20, which prevents prematurepenetration of the solution to be filtered into the filter, or leakingof the solution from the device according to the invention.

However, it is also possible to design layer 20 as a porous, hydrophobiclayer. Where the hydrophobic separating layer 23 is arranged aboveseparating layer 20, it is advantageous if the pore size of thisseparating layer is not smaller than that of underlying layer 20. Withthe other configuration, where the hydrophobic separating layer isarranged beneath layer 20, this requirement is less critical.

Preferably, the device has a piston 80, by means of which a pressure canbe build in the hollow body 40 whereby the passage of the sample throughlayer 20 is promoted.

In another preferred embodiment, the device according to the inventionis capable of being combined with other instruments necessary for thepreparation of nucleic acids, as disclosed, e.g., in P 41 39 664.

P 41 27 276 discloses anion exchangers which are embedded in a membrane(3M Empore Membrane). Such systems are commercially available under thedesignation of QIAwell®.

EXAMPLE 1

Synthesis of Diol-Diatomaceous Earth

100 g of diatomaceous earth is mixed with 2,000 ml of 10% glymo(g-glycidooxypropyltrimethoxysilane) in toluene or carbon tetrachlorideand degassed and refluxed for 6 hours.

The epoxy-diatomaceous earth produced is collected by sucking and washedwith toluene and subsequently with methanol and water.

For the preparation of the diol form, the epoxy-diatomaceous earth isrefluxed with 10 mM H₂SO₄/water for 3 h.

The diol-diatomaceous earth produced is thoroughly washed with water anddried.

The dried diol-diatomaceous earth is directly employed for filtration.The diol-diatomaceous earth may also be synthesized according to othersynthesis protocols such as those described in the literature.

EXAMPLE 2

Filter for Fermenter Cultures

A chromatographic column of about 2,000 ml (10 cm×25 cm) is closed atits lower end with an appropriate 50 μm PE frit or a nylon net andfilled with diol-diatomaceous earth to a height of 5 cm. Thediol-diatomaceous earth is covered with a very coarse filter cloth (ca.150 to 200 μm). Two liters of E. coli cell lysate (prepared from thelysis of 1 vol. of E. coli cells in TE with 1 vol. of 0.1 M NaOH/1% SDS,and neutralisation with 1 vol. of 3 M potassium acetate, pH 4.5) isfilled into the filter column and allowed to rest for 15 min. Thefiltrate is pumped off through the lower column opening by means of aperistaltic pump and directly passed through a anion-exchangechromatographic column with DEAE-Sepharose FF (Pharmacia).

EXAMPLE 3

A 15 ml disposable syringe (diameter: 1.5×15 cm) is closed at its lowerend with a porous 50 μm PE frit and filled with 15 to 25 μm of silicagel to a height of 1 cm and again closed at its upper end with a 50 μmfrit according to FIG. 1. A 100 ml E. coli cell culture is centrifugedand resuspended in 5 ml of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) andlysed by the addition of 1 vol. of 0.1 M NaOH/1% SDS. The cell lysis isstopped by the addition of 1 vol. of 3 M potassium acetate, pH 4.8, andthe cell lysate including the precipitated proteins, K-dodecyl sulfate,genomic DNA and cell fragments is filled in the filter column. The celllysate rests for 10 min wherein the lighter cell fragments, proteins andthe precipitated KDS rise to the top. Such incubation prevents prematureclogging of the filter layer with the cell fragments and thus increasesthe soil uptake and retaining capacity of the filter layer by severaltimes its value. The filtrate is pressed through the silica gel layerwith a piston. Subsequently, the clear lysate can immediately besubjected to a further nucleic acid purification by anion exchangechromatography, gel filtration or precipitation with alcohol.

The filter layer with the cell fragments is discarded.

What is claimed is:
 1. A filtration process for the preparation ofplasmid-DNA from a microorganism, the plasmid-DNA being suitable forgene therapy, the process comprising the steps of: (a) lysing themicroorganism, thereby effecting a lysate; (b) allowing the lysate torest for at least one minute; (c) passing the resulting lysate through afilter layer of packed diatomaceous earth that has not been modifiedwith anion exchange groups, having a particle size between 5 μm and 500μm; and (d) collecting the fraction which elutes from the packeddiatomaceous earth, optionally followed by isolating the plasmid-DNAfrom the collected fraction.
 2. The process according to claim 1,wherein the diatomaceous earth is modified to have no affinity fornucleic acids.
 3. The process according to claim 2, wherein thediatomaceous earth is modified with minerals bearing hydroxy groups. 4.The process according to claim 3, wherein the minerals are diol silicagel, diol-diatomaceous earth, and/or diol-perlite.
 5. The processaccording to claim 4, wherein the minerals arc diol-diatomaceous earth.6. The process according to claim 1, wherein passing the lysate throughthe diatomaceous earth is facilitated by applying a positive pressure onthe side of the diatomaceous earth before the passage or a negativepressure on the side of the diatomaceous earth after the passage.
 7. Theprocess according to claim 1, wherein said diatomaceous earth consistsof untreated diatomaceous earth or diol-diatomaceous earth having flowvalues of from 0.1 to 15 Darcy.
 8. The process according to claim 1,wherein said diatomaceous earth is arranged in a hollow body.
 9. Theprocess according to claim 1, wherein said diatomaceous earth isarranged in a hollow body between two fixing means.
 10. The processaccording to claim 1, wherein said diatomaceous earth is arrangcd in asyringe body between two porous means.
 11. The process according toclaim 10, wherein said porous means are glass or plastic frits.
 12. Theprocess according to claim 6, wherein said positive pressure is appliedby means of a piston.
 13. A kit for perfoiming the process according toclaim 1 comprising: (a) a device containing the diatomaceous earth and(b) buffers required for performing the process.
 14. The kit accordingto claim 13, wherein said device is a disposable syringe.
 15. The kitaccording to claim 14, further comprising anion-exchange columnscontaining filters for the preparation of plasmid-DNA and/or cosmid-DNA.